Infectious recombinant AAV are being developed as gene transfer vehicles for an ever-widening array of human applications such as for use as vaccines and gene therapy vectors. The intense interest in rAAV has been fueled by the finding that these simple vectors can efficiently transduce a variety of post-mitotic cells when administered in vivo. Promising data from animal models has resulted in the initiation of several ongoing human clinical trials. While these advances are encouraging, obstacles remain for the general implementation of rAAV as a universal gene transfer vehicle.
Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J. Virol., 45: 555-564 (1983) as corrected by Ruffing et al., J. Gen. Virol., 75: 3385-3392 (1994). Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named for their relative map locations), drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. Rep 78 and Rep 68, are respectively expressed from unspliced and spliced transcripts initiating at the p5 promoter, while Rep 52 and Rep 40, are respectively expressed from unspliced and spliced transcripts initiating at the p19 promoter. Rep proteins possess multiple enzymatic properties which are ultimately responsible for replicating the viral genome. Rep 78 and 68 appear to be involved in AAV DNA replication and in regulating AAV promoters, while Rep 52 and 40 appear to be involved in formation of single-stranded AAV DNA. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).
When wild type AAV infects a human cell in culture, the viral genome can integrate into chromosome 19 resulting in latent infection of the cell. Production of infectious virus does not occur unless the cell is infected with a helper virus (for example, adenovirus or herpesvirus). In the case of adenovirus, genes E1A, E1B, E2A, E4 and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and adenovirus are produced.
AAV possesses unique features that make it attractive for delivering DNA to cells in a clinical application, for example, as a gene therapy vector or an immunization vector. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of AAV-vectors less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
Production of rAAV requires the AAV rep78/68, rep52/40 and capsid genes and expression of their gene products, a DNA of interest flanked by AAV ITRs, helper functions provided by an adenovirus or herpesvirus helper virus, and a cell line comprising these components that is permissive for AAV replication. Examples of helper virus functions are adenovirus genes E1a, E1 b, E2A, E4 and VA RNA [Carter, Adeno-associated virus helper functions in “Handbook of Parvoviruses” Vol I (P. Tjissen, Ed.) CRC Press, Boca Raton, pp 255-282 (1989)]. Wild type AAV (wt AAV) has one of the largest burst sizes of any virus following infection of cells with AAV and adenovirus. This may be well in excess of 100,000 particles per cell [Aitken et al., Hum Gene Therapy, 12:1907-1916 (2001)], while some rAAV production systems have been reported to achieve greater than 103 particles per cell. Rep proteins are absolutely required for both wt AAV and rAAV replication and assembly of intact infectious particles, as summarized in Carter et al., AAV vectors for gene therapy, in “Gene and Cell Therapy: Therapeutic Mechanisms and Strategies”, Second Edition (Ed. N. Templeton-Smith), pp 53-101, Marcel Dekker, New York (2004).
A requirement for the clinical use of recombinant AAV for DNA delivery is a highly efficient scheme for production of infectious recombinant virus that is reproducible and commercially scalable. One popular mechanism of producing rAAV is to transiently transfect cells with one or more plasmids containing adenoviral helper genes, rep and cap genes, and a recombinant AAV genome. Such transfection methods are difficult to scale up, which has lead to development of stable cell line methods.
Two types of stable cell lines have been developed. In one type (producer cells), both the rAAV genome and the rep-cap genes are stably integrated into the cell DNA, while helper functions are provided by a wild-type adenovirus. As used herein, “producer cells” are those cells that are stably transformed with a rAAV genome and AAV rep/cap genes. In the second type (packaging cells), the rep and cap genes are integrated, while the rAAV genome is provided by infection with a recombinant adenovirus or herpes virus containing the rAAV genome (termed herein a “rAd/AAV hybrid” or “rHerpes/AAV hybrid”), and the helper functions are provide by a wild type adenovirus. As used herein, “packaging cells” are those cells that are stably transformed with AAV rep/cap genes.
The most common forms of these scalable systems use HeLa cells. Other cell substrates have also been used to produce AAV. One such cell substrate is a Vero cell. See, for example, U.S. Patent Application US20040224411 published Nov. 11, 2004; Handa et al., Journal of Biological Chemistry 254(14): 6603-6610 (1979); Richardson et al., Proc Natl Acad Sci USA 77(2): 931-935 (1980); and Liu et al., Journal of Virology 80(4): 1672-1679 (2006). Vero cells are derived from African green monkey kidney cells, and were identified as a cell line substrate for viral vector production. Vero cells have been used as a cell line substrate for the production of numerous human vaccines, including poliovirus (both oral and inactivated) and rabies. The safety of the cell line is attested to by pharmacovigilance of more than 20 million doses of rabies vaccine and more than 1 billion of OPV.
Vero cells have been readily adapted for growth in bioreactors on microcarriers and provide consistently high yields of viruses such as polio and rabies viruses. This allows for vaccine purity (less contaminating cell debris), large lots of vaccine (i.e., greater vaccine availability), and more economic production of vaccine. The issues of yield and adaptability to growth in bioreactors are grounds for use of Vero cells that have been provided to the Center for Biologics Evaluation and Research (CBER) division of the FDA by most manufacturers who propose to use them for vaccine production.
There remains a need in the art for new methods for scalable high titer production of rAAV from mammalian nontransformed cancer cells.